Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA)

In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.     

Study of the reaction of grafting acrylamide onto xanthan gum

The present study aimed to study the reaction conditions of grafting of acrylamide on xanthan gum. It was analyzed the influence of reaction conditions, mainly type of initiator activation, initiator concentration and initiator/acrylamide ratio, on graft parameters and copolymer properties. Potassium persulfate was employed as an initiator and heating or N,N,N',N'-tetramethylethylenediamine was used to activate the initiator. Reaction time and initiator concentration were varied and final values for grafting percentage and grafting efficiency were the same for both methods, whereas speed in reaching these values differs from one technique to another. We found that reaction time was inversely proportional to intrinsic viscosity, likely due to main chain degradation promoted by potassium persulfate (KPS); furthermore, the increasing in the KPS concentration lowers grafting percentage, acrylamide conversion and chain degradation, possibly as a result of O(2) formation at high KPS concentrations.     

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.     

Eosinophil cationic protein: overview of biological and genetic features

The eosinophil cationic protein (ECP) is a small polypeptide that originates from activated eosinophil granulocytes. A wide range of stimuli has been shown to induce the secretion of ECP. The gene that encodes the human ECP is located on chromosome 14, and the protein shares the overall three-dimensional structure and the RNase active-site residues with other proteins in the RNase A superfamily. Several single-nucleotide polymorphisms in the human ECP gene have been currently described. ECP has many biological functions, including an immunoregulatory function, the regulation of fibroblast activity, and the induction of mucus secretion in the airway. Additionally, the protein is a potent cytotoxic molecule and has the capacity to kill mammalian and nonmammalian cells. The purpose of this article was to review the known biological and genetic characteristics of ECP that contribute to the understanding of this protein's role in the development and progression of a wide variety of diseases.     

A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction

Abstract: BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B2 receptors (B2R). BbKI was examined on smooth muscle contraction and Ca2+ mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 μm) increased [Ca2+]cand contraction, as observed with BK (2.0 μm). Not blocked by B1 receptors (B1R), the BbKI agonistic effect was blocked by the B2R antagonist, HOE-140 (6 μm), and the involvement of B2R was confirmed in B2R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca2+ release from internal storages, as a consequence of its interaction with B2R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B2R involvement.     

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.     

Structural Characterization and Neuromuscular Activity of a New Lys49 Phospholipase A2 Homologous (Bp-12) Isolated from Bothrops pauloensis Snake Venom

Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on μ-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA₂ from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 ± 6 min (1.4 μM) and 16 ± 6 min (3.6 μM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca²⁺ is replaced by Sr²⁺, being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA₂ family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction.     

Secretome of HepG2 cells infected with dengue virus: implications for pathogenesis

Viral hemorrhagic fever is a clinical syndrome that poses serious global health threat. Among the causative agents, dengue virus (DV) has the highest incidence rate and its infection is the major cause of viral hemorrhagic fever in the world. Although the pathophysiological mechanisms of DV-induced diseases are not yet understood, it is well accepted that liver is a site of viral replication. In this study, we used proteomics to analyze infection of a hepatic cell lineage, HepG2, with DV, focusing on the secreted proteins. 1D-electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were used, allowing the identification of a total of 107 proteins, among which 35 were found only in control secretome and 24 only in infected cells secretome. To validate these data, we performed 2D-eletrophoresis followed by MALDI-TOF/TOF, resulting in the identification of 20 proteins, 8 of them confirming LC-MS/MS results. We discuss the results obtained taking into account the proteins previously described in the secretome of HepG2 cells, proteins present in human plasma and proteins of interest for dengue pathogenesis. Altogether the data presented here provide clues for the progress in the understanding of the role of liver secretion in the progression of the disease.     

Establishment and Partial Characterization of a Continuous Human Malignant Glioma Cell Line: NG97

1. A human glioma cell line, NG97, was established from tissue obtained from a patient diagnosed with a grade III astrocytoma.2. The NG97 cell line has been subcultured for more than 100 passages in standard culture media without feeder layer or collagen coatings.3. NG97 cells grow in vitro as two subpopulations with distinct morphological appearance: stellate cells with pleomorphic nuclei, and small round cells with few processes. The cells have a doubling time of about 72 h and a plating efficiency of 1%. The injection of NG97 cells into congenitally athymic mice induced the formation of solid tumor masses that could be retransplanted every 4 weeks. The cells obtained from tumor mass when cultivated in vitro had a morphology comparable to those of the initial culture.4. This cell line may prove useful for cellular and molecular studies as well as in studies of gliomas treatment.